Metabolite accurate mass measurement

Liquid chromatography—mass spectroscopy has become one of the most powerful analytical techniques in the drug discovery and development proeess. LC tandem MS is obviously the technique of choice for the identification of metabolites as illustrated in this chapter. Exact mass measurements and elemental composition assignment are essential for the characterization of the metabolites. Accurate mass measurements of the product ions, formed in an MS/MS experiment, greatly facilitate the structure elucidation of the metabolites. With the ever evolving technological advances in mass spectrometry and separation science, LC MS will continue to play an important role in metabolite identification in the future. 

By carefully examining the fragmentation pattern of the metabolite and comparison with the mass spectra of the precursor molecule, it is often possible to determine not only the nature of the biotransformation, but also its position in the molecule. In the proceeding example, accurate mass measurement was used to determine that a hydroxyl group had been added to the benzene ring containing the fluorine substituent. 

A slightly different use of accurate mass measurement in drug metabolism studies has been reported [31]. In this investigation, the accurate masses of the ions derived from Glyburide (Figure 5.48) and some of its metabolites were used to calculate the difference in mass, and thus the elemental composition, between certain of the ions observed in the spectra of the various compounds. 

In qualitative analysis, the isotopic distribution remains an important information. For example in the case the parent drug contains Br or Cl, metabolites or decomposition products can be easily identified by considering the isotopic distribution. With accurate mass measurements a list of elemental compositions can be proposed for a compound for a given accuracy range. Because the intensity of the isotopic distribution is also dependent on the elemental composition of the molecule it can be used to reduce the list of possible elemental formulas [17]. 

In tandem MS mode, because the product ions are recorded with the same TOF mass analyzers as in full scan mode, the same high resolution and mass accuracy is obtained. Isolation of the precursor ion can be performed either at unit mass resolution or at 2-3 m/z units for multiply charged ions. Accurate mass measurements of the elemental composition of product ions greatly facilitate spectra interpretation and the main applications are peptide analysis and metabolite identification using electrospray iomzation [68]. In TOF mass analyzers accurate mass determination can be affected by various parameters such as (i) ion intensities, (ii) room temperature or (iii) detector dead time. Interestingly, the mass spectrum can be recalibrated post-acquisition using the mass of a known ion (lock mass). The lock mass can be a cluster ion in full scan mode or the residual precursor ion in the product ion mode. For LC-MS analysis a dual spray (LockSpray) source has been described, which allows the continuous introduction of a reference analyte into the mass spectrometer for improved accurate mass measurements [69]. The versatile precursor ion scan, another specific feature of the triple quadrupole, is maintained in the QqTOF instrument. However, in pre-... 

Advances in high resolution mass analyzers (TOF, FT-ICR, orbitrap) have greatly improved the detection and identification of metabolites based on accurate mass measurements. In single MS mode accurate mass determination is mainly used to differentiate between isobaric ions. Combined with LC-MS, it allows the detection of predicted metabolites by performing extracted ion current profiles... 

A similar approach using accurate mass measurements and predictive fragmentation sofiware was also applied for the examination of the human microsomal metabolism of nefazodone using a linear ion trap-orbitrap hybrid mass spectrometer. Based on a single LC-MS run, using data-dependant acquisition, 15 metabolites of nefazodone could be identified in MS and MS/MS with a mass accuracy better than 3 ppm. 

For spectra interpretation and metabolite characterization accurate mass measurements become a must while it remains complementary to MS", precursor and neutral loss for identifying metabolites in complex biological matrices. 

Hopfgartner, G. Vdbois, F. The impact of accurate mass measurements using quadrupole/time-of-flight mass spectrometry on the characterisation and screening of drug metabolites. Analusis 2000, 28, 906-914. 

In this chapter, we give an overview on how the API techniques work and which factors have an important influence on the performance. Examples are presented mostly from published work to demonstrate how LC-MS, LC-MS-MS, collision-induced dissociations (CIDs), accurate mass measurements and hydro-gen/deuterium exchange have been systematically and successfully applied in the structural elucidation of impurities, degradation products and metabolites. In addition, these also illustrate how mass spectrometry has offered a third dimension to chromatographic method development and validation. 

Recently, Quintela et al. [58] have determined THC and the carboxy metabolite in oral fluid using HPLC coupled to a quadrupole-TOF mass spectrometer. Extreme selectivity of detection and LLOQs of 0.1 and 0.5ng/mL, respectively, were achieved through accurate mass measurement. None of the real positive samples examined was found to contain THC-COOH. 

In many areas of science (chemistry, biochemistry, etc.), and particularly in drug metabolism studies, there is a need to determine the molecular formula of metabolites of interest. This is where the use of MS for accurate mass measurement has transformed the way in which scientists work. Today, it is very easy to obtain an exact mass measurement of a known or unknown substance. 

As shown in Fig. 4.8, the mass difference between the two metabolites is 36.4 mDa and a mass spectrometer with a resolving power of at least 9500 (M/AM = 344/ 0.0364 = 9450) is required to separate between these two metabolites. MS/MS fragmentation combined with accurate mass measurement is the preferred method for structural elucidation of metabolites, especially when it can help to correlate the elemental composition determined in the MS mode. In the example shown in Fig. 4.8, exact mass measurements of the precursor ions are used as lock mass to measure the exact mass of each fragment ion. Exact mass measurements of the fragment ion at m/z 226 help to narrow down the sites of modifications and allows one to distinguish between rabeprazole-sulphide and rabeprazole-aldehyde. 

Figure 5.9. LC-MS (TIC) and UV (220 nm) chromatograms for microsomal incubation of verapamil containing parent molecule and detected metabolites. Data-dependent accurate mass measurements for verapamil, metabolite parents and fragment ions are shown in the inset.

Figure 5.10. Fragmentation pattern for two buspirone metabolites, (a) buspirone- bi-oxide and (b) 5-OH, 4-0-methyl-buspirone, both yielding nominally isobaric fragment ions at m/z 168, which can be correctly assigned with accurate mass measurements.

Superior sensitivity, efficiency, and specificity have made high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the predominant analytical technique for characterization and quantitative analysis of metabolites (Kostiainen et al., 2003 Ma et al., 2006 Prakash et al., 2007). Ion trap, triple-quadrupole, and quadmpole time-of-flight (Q-TOF) mass spectrometers are routinely used to profile and characterize metabolites in plasma and excreta (Ma et al., 2006). The combination of scan types and features available on mass spectrometers of different design (product ion, MS", neutral loss, precursor ion scans, accurate mass measurements) allows identification and characterization of putative and unexpected metabolites with or without little prior knowledge of biotransformation pathways of a given dmg molecule. 

Nevertheless, even with accurate mass measurement by Q-TOF, LTQ-Orbitrap, or LTQ-Fourier transform ion cyclotron resonance (LTQ-FTICR) MS, it is not always possible to fully characterize certain metabolites based solely on mass spectrometric... 

In LC-MS analysis, identification of lipids requires the use of accurate mass measurements for the determination of elemental composition, combined with MS" experiments to get information of the molecular fragments. In addition, in LC-MS, one metabolite may produce multiple peaks due to the presence of isotopes, adducts, and neutral loss fragments. Therefore, ion annotation is crucial in order to recognize a group of ions likely to originate from the same... 

---